Increased Transcription Levels Induce Higher Mutation Rates in a Hypermutating Cell Line
作者： Jürgen Bachl , Chris Carlson , Vanessa Gray-Schopfer , Mark Dessing , Carina Olsson
DOI: 10.4049/JIMMUNOL.166.8.5051
关键词: Point mutation 、 Green fluorescent protein 、 Somatic hypermutation 、 Molecular biology 、 Biology 、 Cytidine deaminase 、 Mutation rate 、 Transgene 、 Trichostatin A 、 Transcription (biology)
摘要: Somatic hypermutation, in addition to V(D)J recombination, is the other major mechanism that generates vast diversity of Ab repertoire. Point mutations are introduced variable region Ig genes at a million-fold higher rate than rest genome. We have used green fluorescent protein (GFP)-based reversion assay determine role transcription mutation hypermutating cell line 18-81. A GFP transgene containing premature stop codon transcribed from inducible tet-on operon. Using promoter enables us study mutability different levels. By analyzing stable transfectants with flow cytometry, can be measured by appearance GFP-positive revertant cells. Here we show correlates its level. Increased levels caused an increased point codon. Treating transfection clone trichostatin A, specific inhibitor histone deacetylase, additional 2-fold increase rate. Finally, using Northern blot analysis activation-induced cytidine deaminase, essential trans-factor for vivo hypermutation mechanism,
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